Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.


Introduction
Emergence and spread of antimicrobial resistance (AMR) are serious global health burden by retarding effectiveness of available antimicrobials used for treatment of bacterial infections [1].Developed against acquired β-lactamase production in Enterobacterales, antimicrobial spectrum of the third-generation cephalosporins (3GCs) has been extended to tolerate the enzymes, such as bla TEM-1 and bla SHV-1 [2].Resistance to 3GCs emerged by production of extended-spectrum β-lactamases (ESBL) that are capable of hydrolyzing 3GCs molecules, which became a major challenge due to its association with multidrug resistance from AMR gene accumulation [3].bla CTX-M , which encodes cefotaximase-Munich (CTX-M) enzyme originated from Kluyvera spp., is the most abundantly distributed ESBL-encoding gene in Enterobacterales and is one of the β-lactamase family that comprises numerous variants [4].To date, 242 variants of CTX-M have been identified and are classified into four groups based on >94% amino acid sequence identity, including CTX-M groups 1, 2, 9, and 8/25 [5,6].bla CTX-M-15 in group 1 and bla CTX-M-14 in group 9 are the most prevalent variants of concern worldwide, including South-East Asia [7].In Thailand, members in groups 1 and 9, including CTX-M-9, -14, -15, -27, -55, and -65, are predominant in human patients, companion animals, swine, and poultry [8][9][10][11][12][13].By its catalytic activity, CTX-M consequently limits clinical efficacy of penicillins, first-generation cephalosporins, and 3GCs that are antimicrobial treatment of choice for infections caused by enterobacteria [14].
Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR-Cas) system that acts as an adaptive immunity to prevent invasion of exogenous genetic materials in bacteria have been applied for genetic engineering biotechnology due to programmability of CRISPR and nuclease activity of Cas proteins.Among several types, CRISPR-Cas9 system that targets double-stranded DNA and subsequently causes DNA cleavage has been introduced for gene editing for reversal of resistance in bacteria to be susceptible to antimicrobial, so-called antimicrobial re-sensitization, as an alternative to antibiotic due to its ease of manipulation [15][16][17].In previous studies, target DNA sequences were selected from regions in antimicrobial resistance gene to design spacer for programming CRISPR-Cas9 function and cleaving the gene that are mostly a single variant, such as bla NDM-1 , bla CTX-M-15 and bla TEM-1 [18][19][20].Kim et al. (2016) revealed the single target sequence of bla TEM and bla SHV for CRISPR-Cas9, but the consensus sequence of all bla CTX-M variants was not detected [21].Selection of the bla CTX-M region as a target for re-sensitization by CRISPR-Cas9 should be an obstacle because of the high DNA polymorphisms.In addition to coding sequence, the common promoter region shared among gene variants is another target of CRISPR-Cas9 for gene inactivation in bacteria [22].For bla CTX-M , ISEcp1 is preferentially associated with major variants that support gene mobilization with transposase and regulation with a strong promoter [23].Nonetheless, inactivation of bla CTX-M by targeting promoter region has never been evidenced to date.
Previous models of CRISPR-Cas9 construction typically targets internal sequences of antimicrobial resistance genes for re-sensitization.For a high number of variants such as bla CTX-M , the spacer is designed to target the specific variant, such as bla CTX-M-15 [24].There is a possibility to broaden the re-sensitization spectrum to encompass more than one variant if spacer sequences are selected based on identification of the prevalent variants from epidemiological information, consensus sequences within bla CTX-M group, and targeting the gene promoter.
Phagemid system, which produces non-replicative bacteriophage particles by the packaging of recombinant phagemid DNA incorporated in viral capsid facilitated by helper plasmids or helper phages.These systems consist of phagemid DNA construction and phagemid particle production.They are feasible tools for DNA delivery into bacterial cells without lytic effect [25].This strategy has been developed and used as a prototype to deliver CRISPR-Cas9 system in a model of E. coli and S. aureus by modification of phagemid vector integrated with CRISPR-Cas9 that cause lethal effects by chromosomal cleavage and antimicrobial re-sensitization by attacking antimicrobial resistance genes [26,27].For a proof-of-concept of programmed CRISPR-Cas system targeting multiple variants of AMR gene, we here constructed CRISPR-Cas9 to eliminate predominant bla CTX-M variants by using two-step cloning process into a phagemid vector for evaluation of CRISPR spacers and for production of non-replicative phage particles (Fig 1).

Bacterial strains and plasmids
E. coli DH5α (ATCC1 68233) was used for plasmid manipulation, and E. coli K-12 wildtype (ATCC1 23716) was used as recipient cells in a re-sensitization model by transformation and transduction in this study.Derivatives of both strains are described in Table 1, and plasmids are present in S1 Table .Competent cells were prepared according to the standard protocol [28].

bla CTX-M target sequence selection and CRISPR construction
Sequences of bla CTX-M-15 and -55 (group 1), bla CTX-M-14, -27, -65, and -90 (group 9) as well as their promoters were initially aligned to observed consensus regions and protospacer adjacent motif (PAM) sequence (NGG) for Cas9 endonuclease by CHOPCHOP web server (http://chopchop.cbu.uib.no)[30,31].Common spacers of each bla CTX-M group and the promoter were selected from both strands that included 20 nucleotides upstream of PAM sequences and had 25-75% GC content without nucleotide polymorphism to be crRNA.In addition, the sequences must not contain off-target sequences on E. coli K-12 genome [32].

Production of FRC319 for delivering CRISPR-Cas9
pHP17_CO7 (helper phage) was modified from pHP17_KO7 [35], bearing a kanamycin resistance (Km R ) selective marker, by inserting a chloramphenicol resistance (Cm R ) gene that interrupted the Km R gene (Fig 4A).The Cm R gene and its promoter (763 bp) were amplified from pCCI by PCR with primers (PvuI_CmR_F and DraIII_CmR_R) and were cloned into pT&A vector before transformation into E. coli DH5α [36].The colonies were selected by Am100 and chloramphenicol (12.5 μg/mL; abbreviated to Cm12.5) and confirmed by PCR.pHP17_KO7 and pT&A-Cm R were separately digested by PvuI and DraIII, and Cm R gene fragment was ligated to the pHP17_KO7 backbone to generate pHP17_CO7 using T4 ligase.The ligated products were transformed into E. coli DH5α followed by screening using PCR.
Phage FRC319-G1_II and FRC319-G9 particles were produced from overnight-grown culture of the co-transformant E. coli DH5α in 2 L of 2× YT medium containing Km15 and Cm12.5.After incubation at 37˚C with 200 rpm agitation for 18 h and removal of bacterial cells, phage particles were precipitated by PEG/NaCl at 4˚C for 24 h, centrifuged at 12,000 xg for 20 min, and resuspended in 20 mL SM buffer before filtration by using a 0.45-μM filter membrane.The particles in filtrate were repeatedly precipitated, centrifuged, and suspended for purification [37].Ten-fold dilutions of phage in SM buffer from 10 −1 to 10 −8 were prepared for phage titration by adding 5 mL of the phage diluents into 10 mL of mid-log phage E. coli K-12 (OD 600 ~1.5-2.0).Transductants were selectively grown and enumerated on LB agar containing Km15, and phage titers were calculated to be transductant forming unit per mL (TFU/mL) [38].

Target sequences on bla CTX-M for CRISPR spacer construction
One target sequence at the promoter region of bla CTX-M having 25% G+C content was found.Multiple target sequences were presented on alignment of the entire bla CTX-M-15 and -55 (group 1) and bla CTX-M-14, -27, -65, and -90 (group 9) sequence.The candidates from each group were first selected based on the highest efficiency score that met the criteria consisting of number of off-targets, base-pairing affinity, base mismatches, self-complementary regions, and G+C content.Due to low ability for re-sensitization of the first candidate sequence of bla CTX-M group 1, the second candidate was therefore selected for bla CTX-M group 1.All 20-bp target sequences were adjacent to the PAM sequence and had no DNA polymorphism, compared to the tested variants and the promoter (S1 Fig) .The DNA sequence of the targets and their characteristics were presented in Table 2. Aligned with the sequence of all variants in each bla CTX-M group in the NCBI Reference Gene Catalog, the candidate target sequence of bla CTX-M group 1 (bla CTX-M group 1_II) and bla CTX-M group 9 were present in 107 of 108 variants (99.07%) of bla CTX-M group 9 and 69 of 71 variants (97.18%) of bla CTX-M group 9.

Target sequences in CRISPR array of pCRISPR and phagemid pRC319
The 341-bp fragments of CRISPR array including inserted oligos were amplified from pCRISPR-G1_I, pCRISPR-G1_II, pCRISPR-G9, and pCRISPR-P, and the nucleotide sequencing revealed presence of the candidate target sequence as a spacer in each modified CRISPR array for expression to be crRNA (S2 Fig) .After cloning into pRC319, 421-bp fragments of CRISPR region and array were amplified from pRC319-G1_I, pRC319-G1_II, pRC319-G9, and pRC319-P followed by sequencing that revealed the presence of spacer that was identical to those found in the modified pCRISPR.

Antimicrobial re-sensitization by transformation
Re-sensitization activity was present in the programmed CRISPR-Cas9 that targeted internal sequence of bla CTX-M group 1 and bla CTX-M group 9 but was not found in that targeting the promoter region.Viable cells were not reduced in controls of all E. coli K-12: bla CTX-M strains treated with pRC319 transformation that resulted in a ratio of viable cells of 1.In the bla CTX-M group 1, the transformants E. coli K-12: bla CTX-M-15 : pRC319-G1_I and E. coli K-12: bla CTX-M-55 : pRC319-G1_I exhibited a 0.

Discussion
Various studies present the ability of programmable CRISPR-Cas9 system for the reversal of AMR bacteria that have corresponding resistance mechanisms mediated by specific antimicrobial resistance genes to be susceptible to the antimicrobials.Our results proposed searching of common candidate sequences for programming CRISPR-Cas9 by construction of spacer to be a programmed Cas9-tracrRNA-crRNA complex that breakdown multiple variants of major bla CTX-M .Regarding high variation of bla CTX-M , only re-sensitization by CRISPR-Cas9 catalyzing most widespread bla CTX-M-15 was studied [24].However, it was not possible to find consensus regions shared between bla CTX-M groups to be candidate spacers because of low nucleotide similarity and lack of adjacent PAM sequence.Therefore, we selected candidate target sequences from each bla CTX-M group that were capable of programming a spectrum of CRISPR-Cas9 functions.Consistent re-sensitization activity of CRISPR-Cas9 among tested variants in each group supported this approach.Promoter or transcription initiation regions of antimicrobial resistance genes are other targets for of CRISPR-Cas9 system for re-sensitization or gene silencing in bacteria and mammalian cells [22,39].Knocking down expression of ADP-ribosyl transferase using CRISPRi-dCas9 targeting the promoter of arr gene exploits successful re-sensitization of Mycobacterium smegmatis to be susceptible to rifampicin [22].The promoter of bla CTX-M , commonly located on the ISEcp1, was selected to be a broad target of bla CTX-M in this study.However, effective re-sensitization was not obtained by targeting this region.Because of the narrow range and presence of only one PAM sequence, the promoter is not the good target for bla CTX-M inactivation.Therefore, the internal sequence of bla CTX-M gene that had a variety of region adjacent to PAM sequences had a possibility for searching good candidate target sequences for CRISPR-Cas9.Re-sensitization activity was observed in the programmed CRISPR-Cas9 with the highest efficacy, the second candidate target sequence of bla CTX-M group 1 and that of bla CTX-M group 9 can be found in up to 99% (107 of 108 variants) and 97% (69 of 71 variants) of the bla CTX-M variants in each group, respectively [6].In vitro efficacy was examined for the prevalent variants that were available for plasmid construction, and further variants should be further evaluated.Target sequence selection for CRISPR construction and production of guide RNA (gRNA) is a critical step for CRISPR-Cas9 re-sensitization efficacy.For bla CTX-M genes, gene classification and epidemiological information are useful for target sequence selection due to the absence of candidate consensus sequence of all bla CTX-M variants.In contrast, common target sequences for CRISPR-Cas9 on bla TEM and bla SHV , which also have high numbers of variants are detected, and selected for spacer design that results in clearance of AMR gene-containing plasmid and 99% re-sensitization in E. coli [21].In bla CTX-M group 1, a new spacer in pRC319-G1_II was selected due to the low re-sensitization efficacy of pRC319-G1_I that might be a result of low gRNA affinity.DNA region adjacent to the target sequence could affect gRNA activity such as polymorphisms in the PAM sequence and flanking regions [40].5 0 -CGG PAM sequence, which was the PAM sequence of pRC319-G1_II and pRC319-G9, had a higher binding affinity to targets than other PAM sequences for CRISPR-Cas9 [41].Additionally, affinity of "seed sequence", which is the 10-12 nucleotides adjacent to PAM, strongly affects base pairing of gRNA to the target sequence that is a key role for CRISPR array designation [42].As presented in target sequence in the bla CTX-M promoter, borderline G+C content at 25% might retard the binding affinity between the gRNA and the promoter region that causes incomplete pairing and loss of double strand break activity [43].However, few spacer sequences were selected for CRISPR construction in this study which was a limitation for comparison of efficacy and specificity.The most effective single spacer for each bla CTX-M group cannot re-sensitized all resistant cells that are similar to results from previous works [27].Improving strategies such as inserting multiple spacers and creating multiple CRISPR-Cas copies could be merged to in CRISPR-Cas9 development [24,44].
Numbers of re-sensitized cells exponentially increased depending on numbers of phagemid particles that presented CRISPR-Cas9 delivery by the non-replicative phagemid transduction.Bacteriophage has been popularly used as a biological vehicle for introduction of exogenous DNA into bacterial cells.Previously developed phagemid pRC319 contained CRISPR-Cas9 components, multiple recognition site, and f1 origin of replication derived from bacteriophage that fully supported genetic modification, evaluation of CRISPR spacers by transformation, and phagemid particle production in our study.Production of phagemid particle required structural capsid proteins from M13-derivative helper plasmid pHP17_CO7 that was modified from pHP17_KO7, which has kanamycin resistance marker (aminoglycoside phosphotransferase), because of the similar antibiotic resistance selective marker with the modified pRC319, which has neomycin/kanamycin resistance marker.Using co-transformation of pRC319 and pHP17_KO7, very low phage particles were obtained, and loss maintenance of pHP17_KO7 was observed.Phage particle production was achieved by helper plasmid modification to be pHP17_CO7 by insertion of chloramphenicol acetyltransferase gene that supported co-selection of both phagemid and helper plasmid in the co-transformant.In phagemid system, phagemid and helper plasmid containing different antimicrobial resistance genes that need different antimicrobial classes for selection supports bacteria to continuously maintain both in culture of phage particle production system [45].Because pHP17_CO7 is a low-copy number plasmid and lacks its packaging signal, the modified phagemid genome could be sufficiently packaged to be a CRISPR-Cas9 carrier [35].However, helper plasmid particles without the modified pRC319 were not investigated.Compatibility of the helper plasmid pHP17_CO7 and the modified pRC319 containing constructed CRISPR-Cas9 system efficiently generated high numbers (10 10 TFU/mL) of phagemid pRC319 particles in the re-sensitization model.
In this study, we used pCRISPR as an intermediate vector that is adaptable for spacer construction and serves next-step recombination with the phagemid pRC319.This vector is prepared for two delivery modes, including transformation for the evaluation of the constructed CRISPR-Cas system and transduction after phage production.The re-sensitization efficacy was limited only in vitro of E. coli K-12 model in this study.Due to the narrow host range of M13-derived phagemid particle, CRISPR-Cas9 might not be delivered into F-pilus-deficient E. coli strains that needs strategies to expand host range.Effective re-sensitization was presented by catalytic activity on predominant bla CTX-M variants in group 1 and group 9 that were available in our study; however, ability to cleavage other less prevalent variants should be further evaluated.The platform used in our study promoted customizing CRISPR-Cas9 construction to target antimicrobial resistance genes for antimicrobial re-sensitization or to inactivate other genes in E. coli in one-direction, two-step process.Spacers of bla CTX-M genes could be selected from internal sequence that are shared among variants in each group.Epidemiological distribution and AMR gene evolution should be continuously monitored together with the development of gene-based tools combatting AMR.Simplification of cloning methods and development of vector backbone are still promising to facilitate effective introduction of CRISPR-Cas9 and expression in bacterial cells.

Conclusion
This study demonstrated the re-sensitization by phagemid particle carrying CRISPR-Cas9 system.The modified CRISPR-Cas9 delivered by phagemid particle effectively destroys major bla CTX-M variants in E. coli resulting in double strand break and loss of entire plasmid.Selection of candidate spacers from the conserved regions in bla CTX-M groups could serve as a model for broadening cleavage activity of CRISPR-Cas9, targeting not only a single AMR gene, but also those with a high number of variants.Consequently, this technique could be further developed as a promising tool for tackling antimicrobial resistance.

Fig 2 .
Fig 2. Schematic presenting workflow of plasmid construction and antimicrobial re-sensitization by transformation.(A) The candidate target sequence was inserted into CRISPR array on pCRISPR, and then CRISPR array was constructed into phagemid pRC319 that contained Cas9 and tracrRNA and the programmed CRISPR-Cas9 targeting bla CTX-M genes and the promoter (B) The modified pRC319 containing the CRISPR-Cas9 system was transformed into E. coli K-12 carrying bla CTX-M on pMBLe plasmid.The programmed CRISPR-Cas9 expressed functional Cas9-tracrRNA-crRNA complex to cleave pMBLe-bla CTX-M plasmid at the target site that resulted in antimicrobial re-sensitization and plasmid clearance.The figure was generated by BioRender web software (www.app.biorender.com).https://doi.org/10.1371/journal.pone.0303555.g002